This application is a continuation-in-part application of copending U.S. Application Ser. No. 438,991, filed Nov. 1, 1982, now abandoned. This application is also related to copending U.S. Application Ser. No. 338,704, filed Jan. 11, 1982.
This application relates to human interferons and their production in Chinese hamster ovary cells and therapeutic formulations including the human interferons so produced.
Interferons (IFNs) are relatively small, species-specific, single chain polypeptides, produced by mammalian cells in response to exposure to a variety of inducers such as viruses, polypeptides, mitogens and the like. They exhibit antiviral, antiproliferative and immunoregulatory properties and are, therefore, of great interest as therapeutic agents in the control of cancer and various other antiviral diseases (J. Desmyter et al., Lancet 11, 645-647 (1976); R. Derynck et al., Nature 287, 193 (1980)). Human IFNs are classified into three major types, fibroepithelial (IFN-.beta.), leukocyte (IFN-.alpha.) and immune (IFN-.gamma.).
Although IFNs have been known for nearly twenty-five years, characterization of the molecules in terms of their structure and properties has been hampered by the paucity of material available for such studies. Naturally occurring or native IFNs have to be isolated and purified from human sources, which is a very time-consuming and expensive process. Clinical studies to demonstrate the use of IFNs as therapeutic agents have, likewise, been severely limited by the small amounts of pure material available.
In recent times, with the advent of recombinant DNA technology, IFN genes have been identified, isolated, cloned and expressed in microorganisms. Several IFN-.alpha. genes have been cloned and expressed in E. coli (Nagata, S., et al., Nature 284:316-320 (1980); Goeddel, D. V., et al., Nature 287:411-415 (1980); Yelverton, E., et al., Nucleic Acids Research, 9:731-741, (1981); Streuli, M., et al., Proc. Natl. Acad. Sci., (USA), 78:2848-2852 (1981).
Similarly, IFN-.beta. gene has been cloned and expressed in E. coli (Taniguchi, et al., Gene 10, 11-15 (1980)).
Although at least some IFNs are believed to be glycoproteins, IFN-.beta. has been shown to be a glycoprotein by chemical measurement of its carbohydrate content. It has one N-glycosidyl attachment site (E. Knight, Jr., Proc. Natl. Acad. Sci., 73, 520 (1976); E. Knight, Jr., and D. Fahey, J. Interferon Res., 2 (3), 421 (1982)). Even though not much is known about the kinds of sugars which make up the carbohydrate moiety of IFN-.beta., it has been shown that the carbohydrate moiety is not essential for its antigenicity, biological activity or hydrophobicity (T. Taniguchi et al., supra; E. Knight, Jr., supra; and E. Knight, Jr. and D. Fahey, supra). E. coli, which is commonly used as a host for the expression of the IFN-.beta. gene, has no mechanism for attachment of carbohydrates to proteins. The IFN-.beta. produced in E. coli by recombinant DNA technology has in vitro anti-viral activity similar to that of native IFN-.beta., indicating that glycosylation is probably not essential for full biological activity. However, studies of E. coli-produced IFN-.beta. suggest that although it retains biological activity similar to that of native human IFN-.beta. even without the glycosyl moieties, it exhibits altered physical properties which may be due in part to the absence of glycosyl residues. For correct characterization of IFNs and for studying of their efficacy as therapeutic agents, it would be desirable to produce them in animal hosts where the protein would be expected to be glycosylated and in the conformation closest to that of native human IFNs. There have been, however, technical problems involved with introducing DNA fragments into animal tissue culture cells which are quite impermeable to nucleic acids. Other problems relating to the production of the host IFN which may be antigenic to other species have had to be addressed and solved, as these samples would not be suitable for clinical and therapeutic uses.
International Patent Application No. PCT/US81/00240, published Sept. 3, 1981, broadly describes processes for inserting DNA into eukaryotic cells and for producing proteinaceous material, but provides no enabling details regarding suitable DNA fragments, hosts, transforming vectors, methods for transformation, promoter and control sequences which facilitate expression, and other essential components.
It would be highly desirable, therefore, to be able to produce human IFNs in mammalian cells, either constitutively or by induction, without the parallel production of the endogenous host IFN.